| Ex vivo Trophoblast-specific Genetic Manipulation Using Lentiviral Delivery
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Author:
Date:
2017-12-20
[Abstract] In this protocol report, we describe a lentiviral gene delivery technique for genetic modification of the rat trophoblast cell lineage. Lentiviral packaged gene constructs can be efficiently and specifically delivered to the trophoblast cell lineage of the blastocyst. The consequences of ‘gain-of-function’ and ‘loss-of-function’ blastocyst manipulations can be evaluated with in vitro outgrowth assays or following transfer to pseudopregnant rats.
[摘要] 在这个协议报告中，我们描述了一种慢病毒基因传递技术的基因修改大鼠滋养细胞谱系。 慢病毒包装的基因构建体可以有效地和特异性地递送至胚泡的滋养层细胞谱系。 “功能获得”和“功能丧失”囊胚操作的后果可以用体外生长测定或转移到假孕大鼠后进行评估。
【背景】胎盘作为母亲和发育中的胎儿之间的通道，对于胎儿生殖是必不可少的（Georgiades et。，<2002>）。它是专门用于促进和协调母体对妊娠和胎儿发育的适应性（Soares等人，2014; Burton等人，2016）。胎盘含有滋养层细胞，它们执行多种特殊功能。滋养层细胞特化的获得需要高度调控的滋养层干细胞和祖细胞群的分化（Maltepe and Fisher，2015）。成熟的大鼠胎盘由两个形态上和功能上不同的区室组成：交界区和迷宫区（Soares等人，2012）。祖细胞，海绵状滋养层细胞，糖原滋养层细胞和多倍体滋养层巨细胞构成交界区。侵入性滋养层谱系来自交界区内的祖细胞（Ain等人，2003; Soares等人，2014）。在妊娠的最后一周，这些细胞移出胎盘并侵入母体子宫隔膜室（Ain等人，2003; ...
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| In vitro Assay for Dendritic Spine Retraction of Hippocampal Neurons with Sparse Labeling
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Author:
Date:
2016-09-20
[Abstract] Dendritic spines are the post-synaptic structures that play a central role in excitatory synaptic transmission. Developmental spinogenesis relies on a variety of stimuli such as those derived from cell-cell communication and their downstream signaling. Here, we describe an in vitro assay of dendritic spine retraction using hippocampal slice culture, in which individual neurons are sparsely and brightly labeled by the Supernova method, for the study of molecular mechanisms of spine development.
[摘要] 树突状棘是突触后结构，在兴奋性突触传播中起重要作用。 发育性的发生依赖于各种刺激物，如来自细胞 - 细胞通讯及其下游信号传导的刺激。 在这里，我们描述了使用海马片段培养的树突状脊柱退缩的体外测定，其中单个神经元被Supernova方法稀疏和明亮地标记，用于研究脊柱发育的分子机制。
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| Labeling of Precursor Granule Cells in the Cerebellum by ex vivo Electroporation
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Author:
Date:
2013-06-20
[Abstract] This protocol will be useful to introduce the genes of interest into the cerebellar granule cells at early stages of development. Since the granule cell precursors are localized in the external granule layer before migration, DNA plasmids can be specifically incorporated into the granule cells by injecting DNA solution into the cerebellar fissures followed by application of electric pulses. This technique can be performed prior to the preparation of either dissociated or organotypic culture, which can be used to study the molecular mechanisms of cell migration, axon elongation and synapstogenesis during development.
[摘要] 该方案可用于在发育的早期阶段将感兴趣的基因引入小脑颗粒细胞。 由于颗粒细胞前体在迁移前位于外部颗粒层中，因此通过将DNA溶液注射到小脑裂隙中，然后施加电脉冲，可将DNA质粒特异性地掺入颗粒细胞中。 该技术可以在制备解离或器官型培养物之前进行，其可以用于研究在发育期间细胞迁移，轴突伸长和突触发生的分子机制。
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